Spinal disc regenerative composition and method of manufacture and use

ABSTRACT

The present invention provides a novel way to replenish the disc using retooled disc compositions to repair degenerative discs. There is no better source of proteoglycans than the actual disc material ( 6 ) itself. To this end, there has been developed a technique to remove the nucleus pulposus and retool the morphology of the nucleus pulposus to create a powder material ( 10 ) that is dry and can be stored at room temperature for long periods of time. This powder ( 10 ) can then be reconstituted with a variety of fluids, the most suitable being normal saline or lactated ringers to form a flowable mixture ( 20 ).

TECHNICAL FIELD

The present invention relates to a spinal disc regenerative compositionand method of manufacture and use.

BACKGROUND OF THE INVENTION

Intervertebral discs are soft and compressible. They are interposedbetween adjacent vertebral body elements of the spine. They act as shockabsorbers for the spine, allowing it to flex, bend, and rotate.Degenerative disc disease can occur throughout the spine, but most oftenoccurs in the discs in the lower back (lumbar region) and the neck(cervical region).

As the process of degeneration continues, micro tears or cracks occur inthe outer layer (annulus fibrosus) of the disc. The jellylike materialinside the disc (nucleus pulposus) may be forced out through the tearsor cracks in the annulus, which causes the disc to bulge, break open(rupture), or break into fragments.

The economic impact of degenerative disc disease is enormous accountingfor a significant morbidity and lost wages.

The physical properties of the disc are the nucleus pulposus which iscomposed of type II collagen and the annulus fibrosis which surroundsthe disc and gives it significant form. The annulus composed of type Icollagen. The nucleus pulposus is largely made up of molecules calledproteoglycans. These proteoglycans have an affinity for water. It isthis retention of water and the stoichiometry of folded molecules thatis responsible for the unique mechanical properties of the disc. Ifthese proteoglycans are depleted, the discs become more rigid and theloss of fluid results in a disc that is thinner and less compliant.Clinically this results in narrowing of the distances between thevertebral elements. This is best seen on magnetic resonance imaging.Typically discs have a bright signal on T2 pulse-weighted sequences andthey are hypointense on corresponding T1 images. This is due to the highfluid content of the discs. As the disc loses fluid i.e. the loss ofproteoglycans, the disc loses its water signal and becomes anhidroticand eventually mineralizes. As a result, these individuals develop thesymptoms in the spine contributable to loss of the normal discarchitecture. As the process of degeneration continues, one developsmicro tears or cracks and fissures in the annulus fibrosis and throughthese cracks and fissures the nucleus pulposus, which is largelygelatinous, may extrude. The extruded disc material may efface the duraand cause significant nerve compression which may result in traumaticneuritic pain and or motor loss. Therefore, once these early changes indisc degeneration are recognized, it may be prudent to replenish thedisc with proteoglycans. Currently, synthetic and artificial substitutesare used to stimulate repair.

SUMMARY OF THE INVENTION

The present invention provides a novel way to replenish the disc. Thesenovel disc compositions may be used to repair degenerative discs. Thereis no better source of proteoglycans than the actual disc materialitself. To this end, a technique has been developed to remove thenucleus pulposus and retool the morphology of the nucleus pulposus tocreate a powder material that is dry and can be stored at roomtemperature for long periods of time. This powder can then bereconstituted with a variety of fluids, the most suitable being normalsaline or lactated ringers solution to form a flowable mixture.

The powder could also be mixed with stem cells that are derived frommarrow, fat, blood, or any other source, even the interspinousligaments. It could be combined with micronized amnion, platelet-richplasma, and a variety of growth factors that can be encapsulated intopharmacologically active microspheres otherwise known as PAMS. Thepowder could also be combined with genetically altered cells thatproduce large amounts of glycosaminoglycans, collagen Type 1 or glucoseto form the flowable mixture. The micronized material when rehydratedhas a high viscosity and allows the rehydrated material to be flowableas injectable through a cannula. This allows the rehydrated material tobe stored in a syringe or other injectable device for insertion into adamaged disc to be treated.

This flowable mixture forms a composite composition between themicronized nucleus pulposus that can then be injected using a syringe orany suitable injection delivery device through a very small cannula assmall as 2 mm into the disc space. This instrument can be insertedpercutaneously into the disc itself during the process of discography.The flowable material of this composite composition is of a sufficientlyhigh viscosity that once hydrated will not necessarily leak out throughthe injection portal or through pre-existing cracks and fissures in theannulus fibrosus. If however these cracks and fissures are substantial,they could be sealed with fibrin glue as part of the procedure ofintroducing the composites.

DEFINITIONS

As used herein and in the claims:

“Cryomill”—The CryoMill is tailored for cryogenic grinding. The grindingjar is continually cooled with liquid nitrogen from the integratedcooling system before and during the grinding process. Thus the sampleis embrittled and the chemical composition is preserved. The liquidnitrogen circulates through the system and is continually replenishedfrom an Autofill system in the exact amount which is required to keepthe temperature at −196° C. Powerful impact ball milling results in aperfect grinding efficiency. The Autofill system avoids direct contactwith LN2 and makes the operation very safe. Its versatility (cryogenic,wet and dry grinding at room temperature) makes the CryoMill the idealgrinder for quantities up to 20 ml. The grinding jar of the CryoMillperforms radial oscillations in a horizontal position. The inertia ofthe grinding balls causes them to impact with high energy on the samplematerial at the rounded ends of the grinding jar and pulverize it. Thegrinding jar is continually cooled with liquid nitrogen from theintegrated cooling system before and during the grinding process.

“Disc Desiccation”—Disc desiccation is an extremely common degenerativechange of intervertebral discs. The incidence climbs with age, and to alarge degree a gradual desiccation is a ‘normal’ part of disc aging. Itresults from replacement of the hydrophilic glycosaminoglycans withinthe nucleus pulposus with fibrocartilage.

“Freeze Drying”—Freeze-drying, also known as lyophilization,lyophilization, or cryodesiccation, is a dehydration process typicallyused to preserve a perishable material or make the material moreconvenient for transport and stable at room temperatures in anappropriate contained or package. Freeze-drying works by freezing thematerial and then reducing the surrounding pressure to allow the frozenwater in the material to sublimate directly from the solid phase to thegas phase.

“Hypothermic Dehydration”—hypothermic dehydration depends on placing theobject at reduced temperatures above freezing point into a high vacuumchamber allowing it to dry to a desired residual moisture level. Theresult is dried tissue without fissures, microscopic ice crystaldistortion and collapse phenomenon.

“Nucleus Pulposus”—Nucleus pulposus is the gel-like substance in themiddle of the spinal disc. It is the remnant of the notochord. Itfunctions to distribute hydraulic pressure in all directions within eachdisc under compressive loads. The nucleus pulposus consists of largevacuolated notochord cells, small chondrocyte-like cells, collagenfibrils, and proteoglycan aggrecans that aggregate through hyaluronicchains. Attached to each aggrecan molecule are the glycosaminoglycan(GAG) chains of chondroitin sulfate and keratan sulfate. Aggrecan isnegatively charged, allowing the nucleus pulposus to attract watermolecules. The amount of water and glycosaminoglycans decreases with ageand degeneration.

“Proteoglycans”—Proteoglycans are proteins that are heavilyglycosylated. The basic proteoglycan unit consists of a “core protein”with one or more covalently attached glycosaminoglycan (GAG) chain(s).The point of attachment is a Ser residue to which the glycosaminoglycanis joined through a tetrasaccharide bridge (e.g. chondroitinsulfate-GlcA-Gal-Gal-Xyl-PROTEIN). The Ser residue is generally in thesequence-Ser-Gly-X-Gly- (where X can be any amino acid residue, butProline), although not every protein with this sequence has an attachedglycosaminoglycan. The chains are long, linear carbohydrate polymersthat are negatively charged under physiological conditions, due to theoccurrence of sulfate and uronic acid groups. Proteoglycans occur in theconnective tissue. Proteoglycans are a major component of the animalextracellular matrix, the “filler” substance existing between cells inan organism. Here they form large complexes, both to otherproteoglycans, to hyaluronan and to fibrous matrix proteins (such ascollagen). They are also involved in binding cations (such as sodium,potassium and calcium) and water, and also regulating the movement ofmolecules through the matrix. Evidence also shows they can affect theactivity and stability of proteins and signaling molecules within thematrix. Individual functions of proteoglycans can be attributed toeither the protein core or the attached GAG chain and serve aslubricants.

BRIEF DESCRIPTION OF THE DRAWINGS

The invention will be described by way of example and with reference tothe accompanying drawings in which:

FIG. 1A is a photo of a spinal segment after being cut from a spinesegment.

FIG. 1B is a photo of a vertebral spine segment wherein the adjacentvertebrae are cut, separated and the disc material removed.

FIG. 2 is a photo of an exemplary cryomill.

FIG. 3 is a photo of freeze dried disc material micronized to a finepowder.

FIG. 4 is a photo showing the rehydrated disc material flowing from asyringe.

DETAILED DESCRIPTION OF THE INVENTION

The actual disc material 6 is a recovered aseptically, preferably, fromhuman cadaver spine segments 2 from approximately T9 to L5 as shown inFIG. 1A. These are done under sterile conditions. The spinal segmentsare immediately transferred to a processing room where the disc isisolated by cutting the junction between the end plate and thecancellous bone maintaining intact endplates of the vertebral body 4above and below so as not to cause extrusion of the disc material asshown in FIG. 1B. The endplates are then removed and the nucleuspulposus is extracted using sharp dissection. The nucleus pulposus isthen aggregated from all of the intervertebral discs for that particularcase and are placed in a freeze drier and or cold desiccator where themoisture is removed to under 5 percent. The freeze dried material isthen placed in aggregate into a cryomill 100 and micronized into a veryfine powder 10 as shown in FIG. 2. Preferably, the mill 100 pulverizesthe freeze dried nucleus pulposus at low temperatures not exceeding 40°C. to prevent material degradation. The micronized material hasparticles sized less than 400 microns. This fine powder 10, as shown inFIGS. 3 and 4, is then placed into a sterile container and can be storedunder vacuum seal for long periods of time at room air. Once the finepowder material 10 is selected for administration, it is rehydratedusing either normal saline, lactated ringers solution, blood, plateletrich plasma, or a combination of the above. It is then injected into thedisc space using a 2-4 mm cannula, the smaller the cannula the better toprevent extrusion of the material out of the disc space followingadministration. Any pre-existing cracks or fissures are then sealed withfibrin glue after administration of the composite material.

The inventor has developed a biochamber whereby a human disc can beplaced in a physiologic environment and loaded biomechanically.Simultaneously, various parameters can be continuously measured such ascellular activity, oxygen tension and glucose depletion.

It is believed a degenerative disc can be recovered and placed in abiological incubator and injected with the rehydrated freeze driednucleus pulposus powder and incubated over a period of time todemonstrate physiologic repair and healing of the disc by increasedmetabolic activity, water retention and improved biomechanical strength.

This exemplary test protocol can be used to confirm the efficacy of thevarious reconstituted rehydrated mixtures proposed herein.

This allows for a unique method of preparing the material composition ofproteoglycan containing nucleus pulposus comprising the steps of:Aseptic recovery of cadaveric spine segments 2, 4 from T9 to L5 (FIGS.1A and 1B); Removal of the discs 6 by cutting between the cancellousbone and vertebral endplate junction; Removing the normal nucleuspulposus; Freeze drying the nucleus pulposus from multiple discsegments; Placing the freeze dried material into a cryomill 100 (FIG.2); Placing the micronized disc material 10 into a sterile container forlater use (FIG. 3).

Additionally, a test procedure may be used to confirm viability of thematerial which includes the step of: mixing the micronized disc material10 with saline, stem cells, micronized amnion, platelet rich plasma,growth factors, PAMS (pharmacologically active microspheres),genetically altered cells that produce glycosaminoglycans. Thisrehydrated mixture 20 can be made a flowable material suitable fordelivery from a nozzle type container such as a syringe. Once thismicronized powder 10 is rehydrated it can be delivered to treat damagedor degenerative disc repair.

The treatment method can include the steps of: injecting the matrixcomposite through a 2-4 mm cannula into the disc space (FIG. 4). Smallerapertures through which this material may be injected may be preferableto limit extrusion of the material out of the disc space.

The spinal disc tissue can be prepared by dehydration at hypothermictemperatures.

Optionally, the disc material could be extracted from spine segments ofprimates or other mammals.

Variations in the present invention are possible in light of thedescription of it provided herein. While certain representativeembodiments and details have been shown for the purpose of illustratingthe subject invention, it will be apparent to those skilled in this artthat various changes and modifications can be made therein withoutdeparting from the scope of the subject invention. It is, therefore, tobe understood that changes can be made in the particular embodimentsdescribed, which will be within the full intended scope of the inventionas defined by the following appended claims.

What is claimed is:
 1. An aqueous spinal disc regenerative compositionsuitable for injection, wherein the aqueous composition comprises: ahydrated composition comprising freeze-dried micronized nucleus pulposusin a fluid, produced by a method consisting of: (i) providing normalhuman cadaveric intervertebral discs; (ii) separating the nucleuspulposus from the annulus fibrosus of the normal human cadavericintervertebral discs; (iii) drying the nucleus pulposus; (iv)micronizing the dried nucleus pulposus by pulverizing in a cryomill at alow temperature into particles sized less than 400 microns; (v)freeze-drying the nucleus pulposus, wherein said freeze-drying occursduring step (iii), between steps (iii) and (iv), and/or after step (iv);and then (vi) mixing the freeze-dried micronized nucleus pulposus withthe fluid thereby producing the hydrated composition.
 2. The aqueousspinal disc regenerative composition of claim 1 wherein the freeze-driedmicronized nucleus pulposus provided for mixing in step (vi) is in theform of a powder.
 3. The aqueous spinal disc regenerative composition ofclaim 1 wherein the micronized freeze-dried nucleus pulposus hasproteoglycan molecules.
 4. The aqueous spinal disc regenerativecomposition of claim 1 wherein the micronized freeze-dried nucleuspulposus has been dried to less than 5% moisture content prior to beingrehydrated.
 5. The aqueous spinal disc regenerative composition of claim1 wherein the aqueous composition further comprises one or more of thefollowing: stem cells, micronized amnion, platelet-rich plasma, growthfactors, genetically altered cells that produce glycosaminoglycans,collagen Type 1 and glucose.
 6. The aqueous spinal disc regenerativecomposition of claim 1 wherein the micronized material of dried nucleuspulposus has water absorbed by the proteoglycan molecules depleted fromthe freeze-dried nucleus pulposus.
 7. An aqueous spinal discregenerative composition suitable for injection, wherein the aqueouscomposition comprises: a hydrated composition comprising freeze-driedmicronized nucleus pulposus in a fluid, produced by a method consistingof: (i) providing normal human cadaveric intervertebral discs; (ii)separating the nucleus pulposus from the annulus fibrosus of the normalhuman cadaveric intervertebral discs; (iii) drying the nucleus pulposus;(iv) micronizing the dried nucleus pulposus by pulverizing in a cryomillat a low temperature into particles sized less than 400 microns; (v)freeze-drying the nucleus pulposus, wherein said freeze-drying occursduring step (iii), between steps (iii) and (iv), and/or after step (iv);(vi) mixing the freeze-dried micronized nucleus pulposus with the fluidthereby producing the hydrated composition; wherein the freeze-driedmicronized nucleus pulposus provided for mixing in step (vi) is in theform of a powder; and mixing one or more of stem cells that are derivedfrom marrow, fat, blood or interspinous ligaments; micronized amnion;platelet-rich plasma; and/or growth factors encapsulated inpharmacologically active microspheres, with the powdered freeze-driedmicronized nucleus pulposus prior to step (vi), or with the hydratedcomposition after step (vi).
 8. The composition of claim 1 wherein thenormal human cadaveric intervertebral discs are intervertebral discsaseptically recovered from cadaveric spine segments from T9 to LS.
 9. Acomposition consisting of: (a) a syringe or injectable device which iscapable of being inserted into a damaged disc to be treated; and (b) ahydrated composition consisting of freeze-dried micronized nucleuspulposus in a fluid, produced by a method consisting of: (i) providingnormal human cadaveric intervertebral discs; (ii) separating the nucleuspulposus from the annulus fibrosus of the normal human cadavericintervertebral discs; (iii) drying the nucleus pulposus; (iv)micronizing the dried nucleus pulposus by placing the dried material ina cryomill at low temperature and pulverizing the dried nucleus pulposusinto particles sized less than 400 microns into a powder; (v)freeze-drying the nucleus pulposus, wherein said freeze-drying occursduring step (iii), between steps (iii) and (iv), and/or after step (iv);and then (vi) mixing the freeze-dried micronized nucleus pulposus withthe fluid, thereby producing the hydrated composition and mixing one ormore of stem cells that are derived from marrow, fat, blood orinterspinous ligaments; micronized amnion; platelet-rich plasma; and/orgrowth factors encapsulated in pharmacologically active microspheres,with the powdered freeze-dried micronized nucleus pulposus prior to step(vi), or with the hydrated composition after step (vi); wherein the (b)hydrated composition with one or more stem cells is contained within the(a) syringe or injectable device; and wherein the (b) hydratedcomposition has a viscosity which permits it to flow through a cannula.